Site-Specific Cross-Linking of In Vitro Synthesized E. coli Preproteins for Investigating Transmembrane Translocation Pathways

A method is described for the preparation and usage of an E. coli cell-free translation system primed to incorporate the commercially available photoreactive analogue of phenyalanine, p Bpa, into newly synthesized proteins. Incorporation is achieved by means of an amber suppressor tRNA specifically charged with p Bpa. The method is exemplified for the site-specific photocross-linking of the signal sequence of a Tat (twin-arginine translocation) precursor protein to the Tat translocase in the cytoplasmic membrane of E. coli .